Promoter Engineering for Enhanced P(3HB-co-4HB) Production by

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Promoter Engineering for Enhanced P(3HBco-4HB) Production by Halomonas bluephagenesis Rui SHEN, Jin Yin, Jianwen YE, Rui-Juan Xiang, Zhi-Yu Ning, Wu-Zhe Huang, and Guo-Qiang Chen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00102 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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ACS Synthetic Biology

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Title:

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Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas

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bluephagenesis

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Authors:

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Rui Shena1, Jin Yina1, Jian-Wen Yea1, Rui-Juan Xiangc, Zhi-Yu Ninga, Wu-Zhe Huanga,

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Guo-Qiang Chena,b*

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Affiliations:

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a

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for Life Sciences, Tsinghua University, Beijing, 100084, China

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b

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China

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c

Bluepha Co., Ltd., Beijing 102206, China

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Rui Shen, Jin Yin and Jian-Wen Ye contributed equally to this paper

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*

Correspondence:

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Guo-Qiang CHEN

20

School of Life Sciences, Tsinghua University, Beijing 100084, China

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Tel: +86-10-62773844, Fax: +86-10-62788784

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E-mail: [email protected]

MOE Key Lab of Bioinformatics, School of Life Science, Tsinghua-Peking Center

Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084,

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ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract:

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Promoters for the expression of heterologous genes in Halomonas bluephagenesis are

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quite limited and many heterologous promoters function abnormally in this strain.

4

Pporin, a promoter of the strongest expressed protein porin in H. bluephagenesis, is one

5

of the few promoters available for heterologous expression in H. bluephagenesis, yet

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it has a fixed transcriptional activity that cannot be tuned. A stable promoter library

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with a wide range of activities is urgently needed. This study reports an approach to

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construct a promoter library based on the Pporin core region, namely, from the -35 box

9

to the transcription start site, a spacer and an insulator. Saturation mutagenesis was

10

conducted inside the promoter core region to significantly increase the diversity

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within the promoter library. The promoter library worked in both E. coli and H.

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bluephagenesis, covering a wide range of relative transcriptional strengths from 40 to

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140,000. The library is therefore suitable for the transcription of many different

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heterologous genes, serving as a platform for protein expression and fine-tuned

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metabolic engineering of H. bluephagenesis TD01 and its derivative strains. H.

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bluephagenesis strains harboring the orfZ gene encoding 4HB-CoA transferase driven

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by selected promoters from the library were constructed, the best one produced over

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100 g/L cell dry weight containing 80% poly(3-hydroxybutyrate-co-11mol%

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4-hydroxybutyrate) with a productivity of 1.59 g/L/h after 50 h growth under

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non-sterile fed-batch conditions. This strain was found the best for P3HB-co-4HB

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production in the laboratory scale.

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Keywords:

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Promoter library, Halomonas bluephagenesis, polyhydroxyalkanoate, PHB, promoter

25

engineering

26 27 28

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Microorganisms have been used for the production of various goods for humans from

2

ancient times to the present1-4, especially in the fermentative production of enzymes5,

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antibiotics6 and other drugs7, amino-acids8, biofuels9 and biomaterials10. Our main

4

concern, nowadays, is to modify these industrial microorganisms to achieve higher

5

fermentation rates, lower production costs or to generate new products11-13 through

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metabolic engineering or synthetic biology14,

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efficiency of gene expression and the rate of material transport, are key points of

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engineering16-18. Therefore, construction of stable and effective expression systems is

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one of the strategies to improve strains19-21. Genetic tools such as promoters, though

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highly developed in model organisms with clear genetic background such as E.

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coli22-24, often does not work in non-model organisms25-28. The strength

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(transcriptional activity) of a promoter could vary significantly among various

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microorganisms, necessitating the construction of promoter libraries covering a wide

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range of strengths in different model organisms, followed by testing in non-model

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organisms29. On the other hand, the optimization of fermentation conditions offers a

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direct approach to increase productivity30, and should consequently also be taken into

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consideration.

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Halomonas bluephagenesis TD01 and its derivative strains are rising stars of the

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fermentation

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polyhydroxyalkanoates (PHAs), a family of biodegradable plastics, using a low-cost

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non-sterile continuous fermentation process. However, there is still much room for

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improvement. The genetic toolbox for H. bluephagenesis is still at a primary stage,

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lacking a collection of suitable stable promoters32. Efforts have been made to directly

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use promoters from E. coli in H. bluephagenesis, yet the results are unpredictable due

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to the unique genetic background and transcription machinery of H. bluephagenesis.

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A promoter library should be constructed for E. coli, followed by testing with H.

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bluephagenesis. To engineering promoters, the core region from the -35 box to the

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transcription initiation site of promoter Pporin could be modified or removed and

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flanked with the RiboJ33, 34 insulator on the downstream and a spacer on the upstream

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end. Subsequently, mutations could be into the core region forming the promoter

industry31.

They

have

15

been

. To increase the growth rate, the

used

to

efficiently

produce

3



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library (Fig. 1).

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Polyhydroxyalkanoates (PHA) are biodegradable and biocompatible polyesters

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synthesized

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Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB)37 is a PHA with

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desirable properties for many applications, a P(3HB-co-10 mol% 4HB) film was

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shown to be biodegradable in an activated sludge38. H. bluephagenesis TD40 has been

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reported to produce over 70 g/L CDW containing 63% P(3HB-co-12mol% 4HB) after

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48 h growth under non-sterile fed-batch cultivation conditions39. However, this strain

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has only one copy of the Ptac-orfZ construct, with orfZ encoding 4HB-CoA transferase

10

under the control of the tac promoter, integrated into the genome, and its productivity

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can not meet the demands of industry.

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In this study, P(3HB-co-4HB) was tested as a final product generated when orfZ is

13

transcribed under various promoters from the constitutive promoter library. The

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parental strain used in this study is H. bluephagenesis TDhigh, which has the ability to

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achieve high-level PHB accumulation29. H. bluephagenesis TDhigh harbors one copy

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of the PMmP1-LacO-phaCAB module and an inducible Mmp1 system22. The problems

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that need to be solved after inserting the orfZ promoter into the genome of H.

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bluephagenesis TDhigh include the amount and timing of the addition of the inducer

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IPTG and the substrate γ-butyrolactone. The first problem is related to the 3HB mol%

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and toxicity of Mmp1 polymerase29, while the second problem is related to the

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toxicity of γ-butyrolactone40. A series of shake-flask studies were designed and

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conducted to optimize the culture conditions, followed by further adjustments when

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the process was scaled up to a 7 L fermentor studies.

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This study aimed to construct a stable constitutive promoter library with different

25

strengths for both E. coli and H. bluephagenesis to fine-tune heterologous gene

26

expression in H. bluephagenesis. Combined with the optimization of culture

27

conditions, the suitable recombinant H. bluephagenesis would be selected and studied

28

for PHA production.

by

many

microorganisms,

including

H.

bluephagenesis35,

36

.

29

4


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Results and Discussion

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Construction and characterization of a promoter library for E. coli

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Different library construction methods can be involved to generate a library including

4

different variables such as promoters, RBS, CDS, terminators, gene order and/or

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plasmid backbones41. In this case, only promoters were involved in the library design

6

and construction process, while RBS, CDS, gene order, terminators and plasmid

7

backbones remained the same.

8

The synthetic promoters in the library are composed of three parts, a spacer, a core

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region and an insulator, from 5’ to 3’, respectively. The spacer is the upstream

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sequence of the promoter ecf11, while the insulator is RiboJ33. The core region is

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derived from the Pporin promoter42, encompassing the sequences from the -35 box to

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the transcription initial site, 43 bp in total. To construct promoter library 1 (Plib1),

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3-nucleotide saturation mutagenesis was performed upstream of the -10 box, while

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4-nucleotide saturation mutagenesis was performed within the -10 box to construct

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promoter library 2 (Plib2) (Table 1). A total of 288 E. coli colonies containing

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pSEVA321-Plib1-sfGFP or pSEVA321-Plib2-sfGFP were DNA sequenced to find the

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successful construction of 95 non-redundant clones, including a positive control

18

No.70 containing the wild-type promoter core region (Table S1). The negative control

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No.96 did not contain a promoter.

20

Flow cytometry was used to determine the fluorescence strength of the 96 constructs.

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The relative fluorescence intensities (FI) covered a range from 102 to 105, providing

22

adequate choices for further experiments.

23 24

Characterization of the promoter library in different bacteria

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H. bluephagenesis TD01 and its derivatives are being developed as industrial strains

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for PHA production39, and this study is an important addition step toward this goal.

27

Therefore, the promoter library was transferred into H. bluephagenesis followed by a

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cross-context comparison43. The cross-context comparison of library activity showed

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that promoter strengths varied by 2-5 orders of magnitude, both in E. coli and H.

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bluephagenesis TD01 (Fig. 2). Remarkably, the promoter strengths in H.

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bluephagenesis TD01 ware highly correlated with those in E. coli, with an R2=0.98.

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This demonstrated that the promoter interactions of the constructed expression system

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were not significantly affected by the transcriptional machinery of the two strains,

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allowing various levels of genetic control in H. bluephagenesis. On the contrary,

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promoters of E. coli can not perform the same in H. bluephagenesis TD (Fig. S1).

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To investigate whether promoters could properly initiate transcription of target gene

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orfZ, RNA levels of plasmid-borne Plib-sfGFP and Plib-orfZ in H. bluephagenesis TD

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were studied, respectively. The RNA levels of orfZ and sfGFP present similar trends

10

between promoters (Fig. S2). Therefore the promoters could be used for following

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PHA synthesis.

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Application of the promoter library to synthesize PHA in H. bluephagenesis

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The orfZ gene was amplified via PCR from plasmid p68orfZ and used to construct the

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plasmid pSEVA321-promoter_lib-orfZ, followed by the construction of a suicide

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plasmid containing the Plib-orfZ cassette. The plasmid was not used directly in the

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production strain because plasmids are often insufficiently stable in long lasting

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growth44. The gene cassette Plib-orfZ was then inserted into the genome of H.

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bluephagenesis TDhigh using a genome integration method45. Since it was not practical

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to test all 95 promoters in the library via genomic integration, representative

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promoters were selected according to their strength, covering an adequate range

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(Table 2). The strength of the promoter upstream of orfZ in TD40 is around 2000, and

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the FI of the negative control is 108. Strain TDH1 was regarded as the negative

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control in the following experiments.

25

Shake-flask pre-experiments were carried out before optimization of the PHA

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accumulation by H. bluephagenesis TDhigh based strains (TDH1-TDH6, Table 2). In

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the pre-experiment, all the strains were cultured in 50MMG medium with 7.5 g/L

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γ-butyrolactone added at an OD600 of 6 (8 h after inoculation) without an inducer. In

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line with expectations, the promoter strength increased from TDH1 to TDH3, the

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CDW and 4HB mol% also increased. A higher promoter strength indicates a higher 6


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transcription efficiency, resulting in more 4HB being accumulated. The toxicity of

2

γ-butyrolactone has an influence on cell mass, which is why the CDW declined when

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orfZ expression was not adequate. No significant differences were observed among

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the strains H. bluephagenesis TDH3 to TDH6, indicating that a promoter strength of

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700 is sufficient for balancing the toxicity and consumption of γ-butyrolactone.

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However, the promoter was only one of the various factors that influence the

7

synthesis of P(3HB-co-4HB). Other factors include γ-butyrolactone concentration,

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inducer, culture medium, temperature, pH and dissolved oxygen46, 47.

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PHA production by recombinant H. bluephagenesis with optimized promoter

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The γ-butyrolactone, a 4HB precursor, was added at different times and amounts

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(concentrations) in a two-factor experiment (Fig. 3E). H. bluephagenesis TDH4

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consumed less γ-butyrolactone yet was grown to a higher cell dry weight (CDW) (Fig.

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3A). TDH4 consumed more γ-butyrolactone for higher 4HB mol% (Fig. 3B). To

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balance cell growth (CDW) and 4HB mol%, medium amounts of the precursor should

16

be added for further optimization. Regardless of amounts, addition of γ-butyrolactone

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at an OD600 of 6, namely, approximately 8 h after inoculation, led to the highest

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P(3HB-co-4HB)% (Fig. 3C). Accordingly, 5 g/L γ-butyrolactone was added at an

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OD600 of 6 for further inducer optimization.

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To simplify the optimization process, the induction time was first fixed and the IPTG

21

concentration was the only independent variable. IPTG was added at the same time as

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γ-butyrolactone. H. bluephagenesis strains TDH3, TDH4, TDH5 and TDH6 were

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employed since there was no need to optimize TDH1 and TDH2. Interestingly, the

24

results indicated that the CDW of the test group without IPTG induction was

25

significantly higher than that of those with IPTG, regardless of the IPTG amount (Fig.

26

S3).

27

(poly-3-hydroxybutyrate) accumulation, which was the reason of the difference on

28

CDW. However, the transcription of the phaCAB operon should theoretically increase

29

with the addition of IPTG, thus increasing the accumulation of PHB (Fig. S4). The

30

unexpected result may be attributed to synergistic stress brought on by IPTG and

The

IPTG-induced

group

had

a

lower

cell

mass

and

PHB

7



Page 8 of 31

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γ-butyrolactone, both can negatively affect bacterial growth.

2

Since the simultaneous addition of IPTG and γ-butyrolactone yielded suboptimal

3

results, these two compounds should be provided separately. Another round of

4

shake-flask studies was carried out on H. bluephagenesis TDH4. The results

5

demonstrated that IPTG induction at the beginning and at 6 h led to a higher 4HB

6

mol%, respectively (Fig. 4). However, induction at 6 h led to a reduced CDW. The

7

initial induction therefore yielded the best results.

8

The aim of the optimization was to achieve higher PHA content than current industrial

9

strain H. bluephagenesis TD4031. The final flask study was carried out 3 times on

10

three different days. Results revealed that the TCM values (true cell mass,

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TCM=CDW minus P(3HB-co-4HB) weight) of the four experimental strains (H.

12

bluephagenesis strains TDH3, TDH4, TDH5 and TDH6) were similar to that of the

13

control group H. bluephagenesis TD40, indicating that the growth status of the

14

bacteria was very similar, thus the comparison of PHA accumulation was reliable (Fig.

15

5). All four experimental H. bluephagenesis strains performed better in terms of total

16

PHA, PHB and P4HB content, as well as PHA% in CDW. Remarkably, the 4HB

17

content (g/L) of H. bluephagenesis TDH4 was significantly higher than that of H.

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bluephagenesis TD40 when γ-butyrolactone was added at an OD600 of 12, namely,

19

12 h after inoculation (close to the stationary phase). While the 4HB amount (g/L) of

20

H. bluephagenesis TDH5 was significantly higher than that of H. bluephagenesis

21

TD40 when γ-butyrolactone was added at an OD600 of 6 (8 h after inoculation),

22

indicating that these two strains synthesized 4HB from γ-butyrolactone more

23

efficiently. The total PHA content of H. bluephagenesis TDH4 and TDH5 were

24

significantly higher than TD40. Consequently, H. bluephagenesis TDH4 and TDH5

25

were selected for scale-up studies in a 7 L lab fermentor.

26 27

Performance of H. bluephagenesis with optimized promoter in a 7 L fermentor

28

H. bluephagenesis TDH4, TDH5 and TD40 were individually cultured for high cell

29

density growth in a 7 L fermentor using same culture medium and same feeding

30

solution, except that H. bluephagenesis TD40 needs no induction while H. 8


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bluephagenesis TDH4 and TDH5 were induced at inoculation. H. bluephagenesis

2

TDH4 consumed 30 mL γ-butyrolactone while H. bluephagenesis TDH5 and TD40

3

36.5 mL. The feeding strategy was based on a strict control of the residual glucose at

4

10-15 g/L, and the 4 feeding solutions were added sequentially. At the end of the 48 h

5

study, H. bluephagenesis TD40 produced 72 g/L CDW containing 70.0%

6

P(3HB-co-11 mol% 4HB) with a productivity of 1.04 g/L/h (Fig. 6), similar to an

7

earlier study39. While H. bluephagenesis TDH4 was grown to 100.3 g/L CDW

8

containing 79.5% P(3HB-co-11 mol% 4HB) after 50 h, with a productivity of 1.59

9

g/L/h. Notably, this was almost 60% higher than that of the industrial strain H.

10

bluephagenesis TD4039. At the same time, H. bluephagenesis TDH4 consumes 17%

11

less γ-butyrolactone than H. bluephagenesis TD40, yet achieving the same 4HB

12

mol%. H. bluephagenesis TDH5 generated 72 g/L CDW containing 76.4%

13

P(3HB-co-15 mol% 4HB) in 48 h, with a productivity of 1.15 g/L/h, which was

14

almost 10% higher than that of H. bluephagenesis TD40. Remarkably, the 4HB mol%

15

harvested in H. bluephagenesis TDH5 was 40% higher than that of H. bluephagenesis

16

TD40, although H. bluephagenesis TDH5 consumed the same amount of

17

γ-butyrolactone as H. bluephagenesis TD40 did, indicating a higher substrate

18

conversion rate for H. bluephagenesis TDH5.

19 20

Conclusions

21

In this study, the Plib promoter library composed of a spacer, an insulator and a

22

mutated Pporin core origin was constructed. Either 3-nucleotide or 4-nucleotide

23

saturation mutagenesis was conducted in the core region. A total of 288 potential

24

mutants were DNA sequenced, yielding 94 non-redundant mutants. These mutants

25

cloned into an sfGFP reporter construct were grown and generated GFP fluorescence

26

which was measured as a proxy for the transcriptional activity of Plib. In both E. coli

27

and H. bluephagenesis, the FI covered a range from 102 to 105, and a cross-context

28

correlation graph showed an excellent goodness of fit of R2=0.98, indicating that a

29

stable constitutive promoter library was successfully constructed in E. coli and H.

9



Page 10 of 31

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bluephagenesis strains, for fine-tuning heterologous gene expression.

2

Six promoters with strengths of 98.8 ± 26.6, 354 ± 35, 772.7 ± 173.0, 3377.3 ± 378.4,

3

8238.3 ± 1005.1 and 20333.3 ± 1470.5, respectively, were used to chromosomally

4

express orfZ in TDhigh. Shake flask studies demonstrated that the resulting strains H.

5

bluephagenesis TDH3, TDH4, TDH5 and TDH6 all performed better in terms of

6

CDW, total PHA content, PHB content, P4HB content and PHA% than the industrial

7

production strain H. bluephagenesis TD40. Remarkably, the 4HB mol% of H.

8

bluephagenesis TDH4 was significantly higher than that of H. bluephagenesis TD40

9

when γ-butyrolactone was added at an OD600 of 12, while the 4HB mol% of H.

10

bluephagenesis TDH5 was much more than that of TD40 when γ-butyrolactone was

11

added at an OD600 of 6, indicating these two strains synthesized 4HB from

12

γ-butyrolactone more efficiently.

13

Fermrntor studies demonstrated that H. bluephagenesis TDH4 is a stable strain with a

14

high P(3HB-co-4HB) productivity of 1.59 g/L/h, accumulating a similar 4HB mol%

15

as H. bluephagenesis TD40 when fed with 17% less γ-butyrolactone. On the other

16

hand, H. bluephagenesis TDH5 accumulated 40% more 4HB mol% than H.

17

bluephagenesis TD40 when provided with the same amount γ-butyrolactone.

18

H. bluephagenesis TDH4 achieved the highest P3HB-co-4HB production on the

19

laboratory scale to date, demonstrating the significance of a promoter library for

20

engineering industrial strains.

21 22

Materials and Methods

23

Bacterial strains, media and chemicals

24

E. coli S17-148 was used for molecular cloning and plasmid propagation throughout

25

this study. E. coli S17-1, H. bluephagenesis TD01 (collection No.CGMCC4353) and

26

H. bluephagenesis TDhigh29 were used for the characterization of the promoter library.

27

Luria–Bertani (LB) medium: (10 g/L tryptone (Oxoid Ltd., UK), 5 g/L yeast extract

28

(Oxoid Ltd., UK), and 10 g/L NaCl (Sinopharm Chemical Reagent Co., Ltd., China),

29

pH 7.0-7.2) was used for culturing E. coli, while 60LB medium (10 g/L tryptone, 5

10


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1

g/L yeast extract, and 60 g/L NaCl, pH 8.5-9.0) and 50MM medium (minimal

2

medium36 with 50 g/L NaCl, pH 8.5-9.0) were employed for H. bluephagenesis TD01,

3

and LB20 medium (g/L: 10 tryptone, 5 yeast extract, and 20 NaCl, pH 7.0-7.2) plates

4

were used for conjugation (see below). For PHA production, LB and 50MM media

5

with 30 g/L glucose (hereafter “LBG” and “50MMG”, respectively) were used.

6

Chloramphenicol (25 mg/L; Beijing BioDee Biotechnology Co., Ltd., China),

7

spectinomycin (100 mg/L; Beijing BioDee Biotechnology Co., Ltd., China),

8

ampicillin (100 mg/L; Beijing BioDee Biotechnology Co., Ltd., China) or kanamycin

9

(50 mg/L; Beijing BioDee Biotechnology Co., Ltd., China) were added as needed.

10

Isopropyl β-D-1-thio-galactopyranoside (IPTG; AMRESCO Inc., US) was used as

11

inducer. All bacterial strains and plasmids used in this study are listed in Table 3.

12 13

Plasmid library construction and conjugation

14

The promoter Pori sequence (Table 1) was synthesized by Synbio Technologies LLC,

15

China. Its core region, from the -35 box to the transcription initiation site, contains a

16

BsaI restriction site at each terminus, which enable the efficient replacement of the

17

core region, thus introducing variety into the promoters. The low-copy-number

18

plasmid pSEVA32149 with the gene sfGFP was used as vector backbone.

19

pSEVA321-Pori-back was assembled from PCR products of the promoter sequence

20

and vector backbone using the Gibson method50. pSEVA321-Pori-back was the basis

21

of the promoter library (Fig. 7).

22

To generate the library in E. coli, two pairs of degenerate primers were used to obtain

23

75 bp non-template PCR products which were cut with BsaI and ligated into

24

pSEVA321-Pori-back using the Golden Gate method51, yielding two plasmid libraries,

25

pSEVA321-Plib1-sfGFP and pSEVA321-Plib2-sfGFP (Table 1).

26

After promoter strength characterization (see below), several promoters were picked

27

out of the whole library. The orfZ gene was cloned from p68orfZ52 and flanking BsaI

28

sites were added to it by PCR. The sfGFP on the plasmid pSEVA321-Plib-sfGFP was

29

replaced by the orfZ gene using the Golden Gate method to form pSEVA321-Plib-orfZ.

30

To transfer the expression system comprising the promoter library into H. 11



Page 12 of 31

1

bluephagenesis TD, we used interspecific conjugation as described before45. The

2

plasmids pSEVA321-Plib1-sfGFP and pSEVA321-Plib2-sfGFP were transferred into H.

3

bluephagenesis TD01 or TDhigh from E. coli S17-1 via conjugation. Briefly, taking

4

wild-type H. bluephagenesis TD01 as an example, H. bluephagenesis TD01 and E.

5

coli S17-1 carrying the plasmid were cultured separately to an OD600 of around 0.8,

6

after which 10 µL of the donor and recipient cell suspensions were dropped on top of

7

each other and on antibiotic-free LB20 plate and co-cultured for at least 6 h, after

8

which the culture mixture was streaked out onto LB60 plates containing ampicillin

9

and chloramphenicol, in which only H. bluephagenesis transconjugants that acquired

10

the plasmid were able to grow. Finally, a single colony was picked from the selection

11

plate and streaked onto LB60 containing chloramphenicol for further study. All the

12

recombinant H. bluephagenesis strains were stored in 25% glycerol at −80 °C.

13 14

Flow cytometry and characterization of promoter strengths

15

The expression levels of the promoter library were assessed via fluorescence intensity

16

(FI) and mRNA level. FI was measured using an LSRII flow cytometer (BD

17

Biosciences, US) with appropriate voltage settings (FITC 480) and gated by forward

18

and side scatter. At least 20 000 cells were recorded for each sample. Cytometry data

19

were processed using FlowJo (v7.6) to obtain the geometric mean of fluorescence53.

20

The samples for flow cytometry were prepared as follows: Seed culture was streaked

21

out on an LB/60LB plate, followed by 2-day incubation at 37°C, after which a single

22

colony was transferred into 1 mL MMG/50MMG in 96-well deep-well plate, followed

23

by incubation at 37°C and 1000 rpm for 12 h in a deep-well plate shaker. The culture

24

was spun-down and re-suspended in PBS (phosphate buffered saline) in 0.5% volume.

25

RNA levels were investigated using a 7500 Fast Real-Time PCR System (Applied

26

Biosystems, US). The Samples for qRT-PCR were prepared as follows: Seed culture

27

was purified on an 60LB plate, followed by 2-day incubation at 37°C, after which a

28

single colony was transferred into 20 mL 50MMG in a 100 mL shake flask, followed

29

by incubation at 37°C and 200 rpm for 8-12 hours. No antibiotics was added to

30

wildtype H. bluephagenesis TD01, while 25 mg/L chloramphenicol was added to 12


Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

incubate strains harboring the plasmids (Fig. S2), respectively. Total RNA was

2

extracted using the RNAprep Pure Bacteria Kit (TIANGEN, China). Reverse

3

transcription was performed using the FastQuant RT Kit (with gDNase) (TIANGEN,

4

China). The qPCR analysis was conducted using the 2×RealStar Green Power

5

Mixture with ROX II (GenStar, China) based on 16S rRNA as an endogenous control.

6

Relative quantities were calculated using the 2-∆∆Ct method54.

7 8

Genome integration

9

For genome integration, the suicide plasmid pRE112-6ISceI was used as it harbors six

10

I-SceI cutting sites which increase the efficiency during the second recombination45.

11

The whole cassette of the orfZ gene controlled by the selected Plib were cloned into

12

pRE112-6ISceI flanked with two 400-bp homologous arms to form pRE112–

13

6ISceI-orfZ. The PCR products were assembled using the Gibson method and

14

electroporated into electrocompetent E. coli S17-1, followed by colony PCR to verify

15

the plasmid construction. The PCR products were confirmed by DNA sequencing by

16

Boshang Biotech, Inc., China, and the E. coli S17-1 strains carrying correct plasmids

17

were then used as donor cells for the conjugation process. As the correct plasmid

18

enters the recipient cell (H. bluephagenesis TDhigh), it targets the integration site on

19

the genome by homologous recombination. The 1st recombination step occurs when

20

the cassette recombined with the chromosome after the suicide plasmid was

21

introduced into the cell. The 2nd recombination is subsequently triggered by the helper

22

plasmid pBBR1MCS1-ISceI, which expresses ISceI endonuclease targeting the ISceI

23

sequence to produce daughter cells that either carry the Plib-orfZ cassette or revert to

24

the original sequence. Colony PCR was then conducted to confirm the positive clones

25

with recombinant DNA on the chromosome (Fig. S5). The selected knock-in mutant

26

was cultured on LB60 plates in the absence of antibiotic for several generations to

27

lose the helper plasmid. The H. bluephagenesis TDhigh with promoter-orfZ integrated

28

on the chromosome were used for the following experiments.

29 30

Shake flask experiment 13



Page 14 of 31

1

Shake flask experiments were conducted in 50MMG medium comprising (g/L): NaCl

2

50, glucose 30, yeast extract 1, NH4Cl 2, MgSO4 0.2, Na2HPO4·12 H2O 9.65,

3

KH2PO4 1.5, 10 mL/L trace element solution I and 1 mL/L trace element solution II.

4

The composition of trace element solution I was (g/L): Fe(III)-NH4-citrate 5, CaCl2 2,

5

and 1 M HCl. The trace element solution II contained (mg/L): ZnSO4·7H2O 100,

6

MnCl2·4H2O 30, H3BO3 300, CoCl2·6H2O 200, CuSO4·5H2O 10, NiCl2·6H2O 20,

7

NaMoO4·2H2O 30, and 1 M HCl. The pH of the culture medium was adjusted to

8

around 8.5-9.0 using 5 M NaOH36.

9

To prepare seed cultures, the strains were first streaked out on LB60 plates from

10

−80°C glycerol stocks and activated at 37°C, after which a single colony was picked

11

and cultured in LB60 liquid medium for 12 h at 37°C and 200 rpm. The resulting seed

12

culture was used to inoculate the 50MMG medium at 5% volume. Cell growth and

13

PHA production were carried out at 37°C for 48 h in a 500 mL shake flask containing

14

50 mL 50MMG medium. The indicated amounts of γ-butyrolactone and/or IPTG were

15

added, as described in detail in the results section.

16 17

Fermentor studies

18

The laboratory fermentation study was conducted in a 7 L fermentor (New Brunswick

19

Scientific Co., Inc, UK) with a working volume of 3 L. The growth temperature

20

during the fermentation was set to 37°C and the pH was constantly maintained at 8.5

21

via automated addition of 5 M NaOH. The impeller agitation speed was increased

22

gradually from 400 to 800 rpm to maintain the dissolved oxygen concentration at

23

levels above 30% within the first few hours after inoculation, and the agitation rate

24

was set at 800 rpm in the subsequent hours. The flow rate of the inlet gas mix was

25

kept at 1 vvm (Lair/min·Lculture) of initial culture broth volume during the entire

26

process time.

27

No sterilization was required during the entire fermentation. Tap water was directly

28

used for the cultivations instead of distilled water

29

suitable for economic industrial production, the 50MMG medium was slightly

30

modified. Yeast extract was replaced by 15 g/L crude corn extract liquid from

55

. To use low cost substrates

14


Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Shandong Baisheng Biotechnology Co., Ltd, China, and the additional nitrogen

2

source comprised 2 g/L urea at the beginning of the fermentation. Three-stage

3

cultivation was carried out using different feeding solutions. Feeding solution I,

4

containing 800 g/L glucose only, was used to stimulate cell growth and PHB

5

accumulation during the first 18 h. Feeding solution II, composed of 800 g/L glucose

6

and 10 g/L urea, was added between 18 h and 32 h. Feeding solution III containing

7

800 g/L was fed after 32 h56. The flow rate of the feeding solution ranged from 20 to

8

125 mL/h based on the glucose concentration in the fermentors, which was strictly

9

controlled between 10 and 15 g/L. The indicated amount of γ-butyrolactone and/or

10

IPTG was added.

11

The addition time of the substrate or inducer strictly adhered to the optical density of

12

the cell suspension, measured at 600 nm using a Biowave DNA UV

13

spectrophotometer (Biochrom, UK).

14 15

PHA analytical methods

16

For PHA analysis of the flask experiments, culture samples of 30 mL were placed in

17

pre-weighed 50-mL test tubes and centrifuged for 15 min at room temperature and

18

10000 × g. The pellet was frozen at −80 °C after being re-suspended in 20 ml water,

19

and the frozen samples were lyophilized for 24 h before cell dry weight was measured.

20

About 40 mg of lyophilized cell powder was esterified at 100 °C for 4 h by adding 2

21

mL of esterifying mixture (3% concentrated 98% sulfuric acid and 1 g/L benzoic acid

22

in methanol) and 2 mL of chloroform. Standard PHB (99.9%; Sigma-Aldrich

23

(Shanghai) Trading Co. Ltd., China) and γ-butyrolactone (99.9%; Shanghai Macklin

24

Biochemical co.,ltd., China) samples were treated the same as references. After

25

methanolysis, PHA content of the samples was assayed on a GC-2014 gas

26

chromatograph (SHIMADZU, Japan) as described previously57.

27 28

Supporting Information

29

The supporting information is available free of charge on the ACS Publication website

15



Page 16 of 31

1

at DOI: XXXXX.

2

Core sequences of the promoter library; Comparison of interspecies activity of E. coli

3

and H. bluephagenesis TD promoter; RNA level of Plib downstream genes; Inducer

4

optimization results of H. bluephagenesis TD strains; Schematic diagram of genome

5

editing on H. bluephagenesis TD.

6 7

Author Contributions

8

R.S., J.Y. and G.Q.C. designed the research. J.Y. and R.J.X. performed the library

9

construction. R.S. and Z.Y.N. performed the shake-flask experiments. R.S., J.W.Y.

10

and W.Z.H. performed the fermentor experiments. R.S., J.Y. and J.W.Y. performed all

11

the other experiments and analyzed all the data. R.S. and G.Q.C. prepared the

12

manuscript.

13 14

Notes

15

The authors declare no competing financial interests.

16 17

Acknowledgements

18

We are grateful for the donation of pSEVA plasmids by Prof. Victor de Lorenzo of

19

CSIC, Spain. This research was financially supported by a grant from Ministry of

20

Sciences and Technology (Grant No. 2016YFB0302504), and grant for a NSFC

21

Sino-French collaborative project (Grant No. 21761132013). National Natural

22

Science Foundation of China (Grant No. 31430003 and 31600072) also support this

23

project.

24 25

References

26 27 28 29

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Page 20 of 31

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1

Table 1. Original promoter and promoter library Promoter Sequence Pori agcggataacaatttcacacagga(←spacer)atgcctccacaccgc tcgtcacatcctggagacctcactggaatcccagtatagactttgacctg ggtctc(RiboJ→)agctgtcaccggatgtgctttccggtctgatgagt ccgtgaggacgaaacagcctctacaaataattttgtttaa Plib1 agcggataacaatttcacacagga(←spacer)atgcctccacaccgc tcgtcacatcctgttgcgttcactggaatcccannntagagtttgacctgc gagca(RiboJ→)agctgtcaccggatgtgctttccggtctgatgagt ccgtgaggacgaaacagcctctacaaataattttgtttaa Plib2 agcggataacaatttcacacagga(←spacer)atgcctccacaccgc tcgtcacatcctgttgcgttcactggaatcccagtatnnnntttgacctgc gagca(RiboJ→)agctgtcaccggatgtgctttccggtctgatgagt ccgtgaggacgaaacagcctctacaaataattttgtttaa

Description 2 BsaI sites (underlined)

n (underlined) could be a, t, c or g n (underlined) could be a, t, c or g

2 3

21



1

Table 2. Picked Plibs and the corresponding TDhigh based strains Strains Plib No. FI of Plib-sfGFP in H. bluephagenesis TDH1 TDH2 TDH3 TDH4 TDH5 TDH6

23 28 32 50 57 65

98.8 ± 26.6 354 ± 35 772.7 ± 173.0 3377.3 ± 378.4 8238.3 ± 1005.1 20333.3 ± 1470.5

Page 22 of 31

Mutated sequence CTCA GCTA TAGA TTT CTA AGCA

2 3

22


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1

Table 3. Strains and plasmids used in this study Strain/Plasmid Description Strains E. coli S17-1 recA; harboring the tra genes of plasmid RP4 in the chromosome, proA, thi-1 H. bluephagenesis A halophilic bacterium isolated TD01 from a salt lake in China, wild type H. bluephagenesis Recombinant H. bluephagenesis TD40 TD01 with Ptac-orfZ integrated into the genome H. bluephagenesis Recombinant H. bluephagenesis high TD TD01 with PMmp1-LacO-phaCAB integrated into the genome H. bluephagenesis Recombinant H. bluephagenesis TDH1 series TDhigh with Plib-orfZ integrated into the genome separately Plasmids pRE112–6ISceI pRE112 derivate with six I-SceI recognition sites, a suicide vector for gene knock out in H. bluephagenesis TD01, CmR p68orfZ pBHR68 harboring orfZ, AmpR pSEVA321-sfGFP RK2 replication origin, oriT, an expression vector in Halomonas TD strain, sfGFP, CmR pBBR1MCS1-ISceI A helper plasmid, pBBR1MCS1 derivate expressing I-SceI endonuclease, CmR, SpeR pSEVA321-Pori-back pSEVA321-sfGFP derivate with promoter replaced by spacer-Pporin-RiboJ pSEVA321-Plib-sfGFP pSEVA321-Plib-back derivate with series 3 bp saturation mutagenesis before the -10 box or 4 bp saturation mutagenesis inside the -10 box in the Pporin core region pSEVA321-Plib-orfZ pSEVA321-Plib-sfGFP derivate series with sfGFP replaced by orfZ pRE112-Plib-orfZ-6ISceI pRE112–6ISceI derivate with series Plib-orfZ cassette and homologous sequences targeting the integration site

Reference (Simon et al. 1983)48

(Tan et al. 2011)36 (Chen et al. 2017)39

(Zhao et al. 2017)29

This study

(Fu et al. 2014)45

(Li et al. 2010)52 (Silva-Rocha et al. 2013)49 (Zhao et al. 2017)29 (Fu et al. 2014)45

This study

This study

This study This study

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Figure 1

Figure 1. Scheme for the integrated optimization of PHA accumulation through genetic and process engineering. A: The promoter library (Plib) is composed of a spacer, Pporin core and an insulator, in sequence. The spacer and insulator enhance the stability of the promoter. A 3-nucleotide saturation mutagenesis locus before the -10 box or a 4-nucleotide saturation mutagenesis locus inside the -10 box greatly increase the variation within Plib; B: pSEVA321 plasmids harboring Plib and sfGFP were assembled to characterize the promoter strength (transcriptional activity) by flow cytometry; C: TDhigh with selected Plib-orfZ cassettes integrated into the genome (strain TDH series) went through shake flask experiments to optimize the fermentation conditions. D: Strains of the H. bluephagenesis TDH series were cultured in a 7 L fermentor to test their PHA productivity.

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Figure 2

Figure 2. Comparison of transcriptional activity of promoter mutants in E. coli and H. bluephagenesis, respectively. A: Fluorescence intensity (FI) of plasmid-borne Plib-sfGFP in E. coli and H. bluephagenesis or corresponding mutated sequences. B: Positive correlation between the FI of plasmid-borne Plib-sfGFP in E. coli and H. bluephagenesis measured by flow cytometry, respectively. a.u.: arbitrary unit. Error bars represent standard deviations; n=3 biological replicates.

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Figure 3

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Figure 3. 2D design of experiment for optimization of γ-butyrolactone addition in Halomonas bluephagenesis TDH4. A: Cell dry weight. B: 4HB mol%. C: P(3HB-co-4HB)%. D: True cell mass. E: Arrangement table of γ-butyrolactone addition. 2D represents two dimensions: timing and amount of γ-butyrolactone added to the culture. Fermentation conditions: 50 mL culture in 500 mL shake flask, 37 °C, 200 rpm, 50MMG medium without IPTG. γ-butyrolactone was added as indicated in the arrangement table. Error bars represent standard deviations; n=3 biological replicates.

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Figure 4

Figure 4. PHA accumulation by Halomonas bluephagenesis TDH4 with IPTG and γ-butyrolactone added respectively. A: Cell dry weight and true cell mass. B: P(3HB-co-4HB)% and 4HB mol%. Conditions: 50 mL culture in 500 mL shake flask, 37 °C, 200 rpm, 50MMG medium with 5 g/L γ-butyrolactone added at 8 h. IPTG was added to a final concentration of 10 mg/L at 0, 2, 4, 6 and 8 h, respectively (OD600 at 0h=0.3, 2h=0.4, 4h=2, 6h=3.5, 8h=6). TCM indicates true cell mass. TCM=CDW - P(3HB-co-4HB) content. Error bars represent standard deviations; n=3 biological replicates.

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Figure 5. PHA accumulation by Halomonas bluephagenesis TDH strains and H. bluephagenesis TD40, respectively. A: Cell dry weight and true cell mass. B: P(3HB-co-4HB)% and 4HB mol%. C: P(3HB-co-4HB), 3HB and 4HB content. Fermentation conditions: 50 mL culture in 500 mL shake flask, 37 °C, 200 rpm, 50MMG medium with 10 mg/L IPTG added at OD600=2. γ-butyrolactone was added at OD600=6 or 12, respectively. Error bars represent standard deviations; n=9 biological replicates. 28


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Figure 6. PHA production by Halomonas bluephagenesis TDH4, TDH5 and TD40 in 7 L fermentors, respectively. A: Cell dry weight. B. P(3HB-co-4HB)%. C. 4HB mol%. The culture medium and feeding strategy are described in detail in the results section. IPTG was added at 0 h (OD600=2.5), γ-butyrolactone was added when the OD600 exceeded 120 (for TDH4 and TDH5, the time is 16 h after inoculation; for TD40, it is 20 h). 36.5 mL γ-butyrolactone was fed to H. bluephagenesis TD40 and TDH5, while 30 mL was fed to H. bluephagenesis TDH4. Error bars represent standard deviations; n=3 technical replicates

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Figure 7

Figure 7. Schematic diagram of the key features of the plasmid pSEVA321-Pori-back. Plasmid pSEVA321-Pori-back is composed of the P321-sfGFP vector backbone and Pori. Two BsaI sites enable facile replacement of the core region. sfGFP can be replaced by the orfZ gene to generate a cassette for genomic integration.

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For Table of Contents Use Only

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Title: Promoter Engineering for Enhanced P(3HB-co-4HB) Production in Halomonas bluephagenesis Authors: Rui Shen, Jin Yin, Jian-Wen Ye, Rui-Juan Xiang, Zhi-Yu Ning, Wu-Zhe Huang, Guo-Qiang Chen*

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Promoter Engineering for Enhanced P(3HB-co-4HB) Production by

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